Wei-Pang Huang

Mechanism of Cargo Selection in the Cytoplasm to Vacuole Targeting Pathway

The proper functioning of eukaryotic organelles is largely dependent on the specific packaging of cargo proteins within transient delivery vesicles. The cytoplasm to vacuole targeting (Cvt) pathway is an autophagy-related trafficking pathway whose cargo proteins, aminopeptidase I and alpha-mannosidase, are selectively transported from the cytoplasm to the lysosome-like vacuole in yeast. This study elucidates a molecular mechanism for cargo specificity in this pathway involving four discrete steps. The Cvt19 receptor plays a central role in this process: distinct domains in Cvt19 recognize oligomerized cargo proteins and link them to the vesicle formation machinery via interaction with Cvt9 and Aut7. Because autophagy is the primary mechanism for organellar turnover, these results offer insights into physiological processes that are critical in cellular homeostasis, including specific packaging of damaged or superfluous organelles for lysosomal delivery and breakdown.

Autophagy protects neuron from Aβ-induced cytotoxicity

Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimers disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ25-35, Aβ1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.

Nuclear translocation of epidermal growth factor receptor by Akt-dependent phosphorylation enhances breast cancer-resistant protein expression in gefitinib-resistant cells

Epidermal growth factor receptor (EGFR), an aberrantly overexpressed or activated receptor-tyrosine kinase in many cancers, plays a pivotal role in cancer progression and has been an attractive target for cancer therapy. Gefitinib and erlotinib, two EGFR-tyrosine kinase inhibitors, have been approved for non-small cell lung cancer. However, durable clinical efficacy of these EGFR inhibitors is severely limited by the emergence of acquired resistance. For example, the expression of breast cancer-resistant protein (BCRP/ABCG2) has been shown to confer acquired resistance of wild-type EGFR (wtEGFR)-expressing cancer cells to gefitinib. However, the underlying molecular mechanisms still remain unclear. Here, we show that wtEGFR expression is elevated in the nucleus of acquired gefitinib-resistant cancer cells. Moreover, nuclear translocation of EGFR requires phosphorylation at Ser-229 by Akt. In the nucleus, EGFR then targets the proximal promoter of BCRP/ABCG2 and thereby enhances its gene transcription. The nuclear EGFR-mediated BCRP/ABCG2 expression may contribute at least in part to the acquired resistance of wtEGFR-expressing cancer cells to gefitinib. Our findings shed light on the role of nuclear EGFR in the sensitivity of wtEGFR-expressing cancer cells to EGFR tyrosine kinase inhibitors and also deciphered a putative molecular mechanism contributing to gefitinib resistance through BCRP/ABCG2 expression.

Autophagy: A double-edged sword in Alzheimer's disease

Autophagy is a major protein degradation pathway that is essential for stress-induced and constitutive protein turnover. Accumulated evidence has demonstrated that amyloid-β (Aβ) protein can be generated in autophagic vacuoles, promoting its extracellular deposition in neuritic plaques as the pathological hallmark of Alzheimer’s disease (AD). The molecular machinery for Aβ generation, including APP, APP-C99 and β-/γ-secretases, are all enriched in autophagic vacuoles. The induction of autophagy can be vividly observed in the brain at early stages of sporadic AD and in an AD transgenic mouse model. Accumulated evidence has also demonstrated a neuroprotective role of autophagy in mediating the degradation of aggregated proteins that are causative of various neurodegenerative diseases. Autophagy is thus widely regarded as an intracellular hub for the removal of the detrimental Aβ peptides and Tau aggregates. Nonetheless, compelling data also reveal an unfavorable function of autophagy in facilitating the production of intracellular Aβ. The two faces of autophagy on the homeostasis of Aβ place it in a very unique and intriguing position in AD pathogenesis. This article briefly summarizes seminal discoveries that are shedding new light on the critical and unique roles of autophagy in AD and potential therapeutic approaches against autophagy-elicited AD.

White spot syndrome virus protein ICP11: A histone-binding DNA mimic that disrupts nucleosome assembly.

White spot syndrome virus (WSSV) is a large (≈300 kbp), double-stranded DNA eukaryotic virus that has caused serious disease in crustaceans worldwide. ICP11 is the most highly expressed WSSV nonstructural gene/protein, which strongly suggests its importance in WSSV infection; but until now, its function has remained obscure. We show here that ICP11 acts as a DNA mimic. In crystal, ICP11 formed a polymer of dimers with 2 rows of negatively charged spots that approximated the duplex arrangement of the phosphate groups in DNA. Functionally, ICP11 prevented DNA from binding to histone proteins H2A, H2B, H3, and H2A.x, and in hemocytes from WSSV-infected shrimp, ICP11 colocalized with histone H3 and activated-H2A.x. These observations together suggest that ICP11 might interfere with nucleosome assembly and prevent H2A.x from fulfilling its critical function of repairing DNA double strand breaks. Therefore, ICP11 possesses a functionality that is unique among the handful of presently known DNA mimic proteins.

Receptor protein complexes are in control of autophagy

In autophagic processes a variety of cargos is delivered to the degradative compartment of cells. Recent progress in autophagy research has provided support for the notion that when autophagic processes are operating in selective mode, a receptor protein complex will process the cargo. Here we present a concept of receptor protein complexes as comprising a functional tetrad of components: a ligand, a receptor, a scaffold and an Atg8 family protein. Our current understanding of each of the four components and their interaction in the context of cargo selection are considered in turn.

S1P5 is required for sphingosine 1-phosphate-induced autophagy in human prostate cancer PC-3 cells

Sphingosine 1-phosphate (S1P) is a platelet- and endothelial cell-released lysophospholipid that regulates various cellular functions through activating a specific family of G protein-coupled receptors. Both platelet activation and angiogenesis play important roles in cancer development, implying that cancer cells might encounter a large amount of S1P during these processes. Cancer cells, in the meantime, may experience nutrient deprivation and rely on autophagy for early development. Whether extracellular S1P regulates autophagy remains to be tested. In the present work, we investigated whether autophagy is regulated by S1P in PC-3 cells. Through monitoring the modification patterns of LC3 by Western blotting, we demonstrated that autophagy was induced by exogenously applied S1P in PC-3 cells. This observation was further confirmed by fluorescence microscopy using PC-3 cells stably expressing enhanced green fluorescent protein-LC3. By applying small interfering RNA and dihydro-S1P, S1P(5) activation was found to be involved in this process. Besides, mammalian target of rapamycin signaling was inhibited upon S1P treatment. Taken together, our results suggest that, under serum-starved conditions, S1P further upregulates autophagic activity through S1P(5)-dependent pathways in PC-3 cells.

Platonin induces autophagy-associated cell death in human leukemia cells.

Platonin is a photosensitizer used for photodynamic therapy. In this study, we tested the effect of platonin on human leukemic cells. Treatment with platonin in the dark markedly reduced cell membrane integrity, and induced significant G0/G1 arrest of a panel of human leukemic cell lines, including U937, HL-60, K562, NB4 and THP-1. Development of hypodiploid cells was not evident in these cell lines within 24 h, but was noted in U937, HL-60 and NB4 cells after 24 h. No myeloid differentiation of these cells was noted after 5-day treatment. Intriguingly, exposure of monoblastic U937 cells to platonin caused changes characteristic of autophagy, including appearance of cytoplasmic membranous vacuoles and formation of acidic vesicular organelles (AVO) in more than 95% of cells. The platonin-induced autophagy was accompanied by localization of microtubule-associated protein 1 light chain 3 to autophagosomes. Pretreatment with pancaspase inhibitor Z-VAD-fmk abrogated the platonin-induced hypodiploidity, but had no effect on growth inhibition and formation of AVO, indicating a caspase-independent autophagy-associated cell death. Pretreatment of cells with 3-methyladenine attenuated platonin-mediated growth inhibition and formation of AVO. Platonin augmented the expression of BNIP3 in both U937 and K562 cells, whereas had an opposite effect on phosphorylation of mTOR downstream molecule p70S6K. Platonin, at the condition inducing autophagy, induced the mitochondrial membrane permeation. These results suggest that the platonin is capable of inhibiting growth as well as inducing cell death, mainly autophagy-associated, in leukemic cells via a mitochondria-mediated and caspase-independent pathway. A markedly less viability inhibition was noted to human monocytes, the normal counterpart of these myeloid leukemic cells. Platonin, other than a photodynamic agent, may offer significant promise as a therapeutic agent against leukemia.

Areca nut extract induced oxidative stress and upregulated hypoxia inducing factor leading to autophagy in oral cancer cells

Areca (betel) chewing was tightly linked to oral tumorigenesis in Asians. Areca nut was a recently confirmed group I carcinogen and a popular addictive substance used by Asians. While, the pathogenetic impact of areca on oral epithelial cells was still unclear. This study investigated the association between the induction of autophagy by areca nut extract (ANE) and the molecular regulation underlying this induction in oral cancer cells. Oral cancer cells were treated with ANE to insight the signaling changes underlying phenotypic alterations. The NFκB activation and reactive oxygen species (ROS) genesis were induced by ANE and the NF-κB activation could be the basis of the ROS genesis. Furthermore, p38 activation and upregulation of MKP-1 phosphatase occurred following ANE treatment. These effects can be inhibited by ROS blockers. ANE treatment induced autophagy among oral cancer cells, which was characterized by LC3-II accumulation, genesis of autophagosomes and the appearance of EGFP-LC3 puncta. This induction was mediated through the activation of p38, MKP-1 and HIF-1α. Knockdown of ANE-modulated HIF-1α expression reduced autophagy. Blockage of ANE-induced autophagy increased the proportion of oral cancer cells undergoing apoptotic death. This study identified for the first time that ANE modulates a signaling cascade that induces HIF-1α expression in oral cancer cells. The eventual induction of autophagy was beneficial to cell survival from ANE-induced apoptosis.

Diaphanous-related formin 2 and profilin I are required for gastrulation cell movements.

Intensive cellular movements occur during gastrulation. These cellular movements rely heavily on dynamic actin assembly. Rho with its associated proteins, including the Rho-activated formin, Diaphanous, are key regulators of actin assembly in cellular protrusion and migration. However, the function of Diaphanous in gastrulation cell movements remains unclear. To study the role of Diaphanous in gastrulation, we isolated a partial zebrafish diaphanous-related formin 2 (zdia2) clone with its N-terminal regulatory domains. The GTPase binding domain of zDia2 is highly conserved compared to its mammalian homologues. Using a yeast two-hybrid assay, we showed that zDia2 interacts with constitutively-active RhoA and Cdc42. The zdia2 mRNAs were ubiquitously expressed during early embryonic development in zebrafish as determined by RT-PCR and whole-mount in situ hybridization analyses. Knockdown of zdia2 by antisense morpholino oligonucleotides (MOs) blocked epiboly formation and convergent extension in a dose-dependent manner, whereas ectopic expression of a human mdia gene partially rescued these defects. Time-lapse recording further showed that bleb-like cellular processes of blastoderm marginal deep marginal cells and pseudopod-/filopod-like processes of prechordal plate cells and lateral cells were abolished in the zdia2 morphants. Furthermore, zDia2 acts cell-autonomously since transplanted zdia2-knockdown cells exhibited low protrusive activity with aberrant migration in wild type host embryos. Lastly, co-injection of antisense MOs of zdia2 and zebrafish profilin I (zpfn 1), but not zebrafish profilin II, resulted in a synergistic inhibition of gastrulation cell movements. These results suggest that zDia2 in conjunction with zPfn 1 are required for gastrulation cell movements in zebrafish.